Objective 1: To discover gene expression and cytokine biomarkers in the peripheral blood of children with enthesitis-related arthritis (ERA), an early form of spondyloarthritis, that distinguish it from other forms of juvenile idiopathic arthritis (JIA). Ongoing studies of the major JIA subtypes have resulted in several important observations. For example, clinical descriptors such as the number of joints involved during disease have been used to classify children with JIA as oligoarticular (<5 joints) or polyarticular (&#8805;5 joints). Comparing children whose arthritis began at <6 vs. &#8805;6 years of age revealed an unexpected number of peripheral blood gene expression differences that could not be accounted for by the age of the individual. In early onset disease, there was greater expression of genes related to B cells, and lower expression of genes related to myeloid lineage cells. We identified samples from early vs. late onset oligoarticular JIA with 97% accuracy, and early vs. late onset polyarticular JIA with 89% accuracy, while this was not possible with sJIA or healthy controls. PCA showed that the major classifier of oligoarticular and polyarticular JIA was age at onset, rather than the number of joints involved. These studies suggest that the classification of JIA should take into account age at onset, and may reveal important differences in pathogenesis related to B cell activation in early onset disease that has important implications for therapy. Objective 2: To determine the role of HLA-B27 in the development of SpA in an animal model. Previous work has shown that HLA-B27 misfolding can activate the unfolded protein response (UPR) in cells from HLA-B27 and human beta-2 microglobulin transgenic rats (HLA-B27 transgenic rats). These animals develop spondyloarthritis-like disease and thus serve as an experimental model to explore the role of HLA-B27. We have shown that the IL-23/IL-17 axis is strongly activated in the colon of HLA-B27 transgenic rats, and that HLA-B27 misfolding and UPR activation can result in enhanced induction of the pro-Th17 cytokine, IL-23, suggesting a link between HLA-B27 misfolding and immune dysregulation in this model. The discovery that IL23R polymorphisms are associated with predisposition to ankylosing spondylitis and inflammatory bowel disease in genome-wide association studies, suggests potential mechanistic links between HLA-B27 and Th17 activation. Current efforts are aimed at developing reagents to explore the role of Th17 activation in rat spondyloarthritis, including the production of an anti-rat IL-23 monoclonal antibody. In addition we have recently found greater numbers of IL-17-expressing CD4+ splenocytes in HLA-B27 transgenic rats compared to wild type littermates (2.3-fold), and more splenocytes exhibiting STAT3 phosphorylation in response to IL-23 (2-fold). These studies provide the first evidence that Th17 activation extends beyond the colon tissue in HLA-B27 transgenic rats, and suggest that the IL-23/IL-17 axis may be involved in the systemic inflammation that develops in these animals. Targeting IL-23 may provide may provide an important new therapeutic avenue for spondyloarthritis. Objective 3: To explore the effects of ERAP1 on the immunobiology of HLA-B27. ERAP1/ARTS1 encodes an ER aminopeptidase that is involved in trimming peptides that are destined for presentation by MHC class I molecules. ARTS1 is a product of the same gene, and was discovered independently as an 'aminopeptidase regulator of TNF receptor shedding'. Subsequently, a role in IL-6R and IL-1RII shedding has been demonstrated. Key disease-related polymorphisms, and their potential role in pathogenesis, remains unexplored. We have initiated studies to examine the role of ERAP1 on the immunobiology of HLA-B27 with basic loss-of-function/gain-of-function studies. Since we are able to study the role of HLA-B27 in the transgenic rat model, we are screening rat ERAP1 knockdown constructs (shRNA). This is being done in rat1 fiibroblasts, and human constructs are being screened in HEK293 cells. Two shRNA for rat ERAP1 have been identified that reliably reduce ERAP1 expression on Western blots by 90-95% after 96 hours. Controls include an empty vector and also a scrambled vector construct. We need to confirm reduced expression of ERAP1 mRNA, and are currently screening stable transfectants made with retroviral constructs. Knockdown of human ERAP1 to about 60% of control levels has been achieved in HEK293 cells, but we are still screening for more effective seqeunces. Preparation of cell lines expressing HLA-B27 and other 'control'MHC class I molecules with reduced or absent ERAP1 expression, will enable us to examine steady state levels of various folded and misfolded forms of HLA-B27 as a means of determining how ERAP1 affects the assembly of HLA-B27 complexes. Future experiments using lentivirus-delivered ERAP1 shRNA constructs to HLA-B27 transgenic rat cells will allow us to determine effects on HLA-B27 misfolding during upregulation, and ultimately whether the HLA-B27-induced unfolded protein response is modulated. Similar studies will be performed in human cells. Objective 4: To determine the effects of susceptibility genes on osteoclast and osteoblast development and function. One of the most poorly understood aspects of spondyloarthritis pathogenesis is bone loss (erosions and osteoporosis) juxtaposed with abnormal bone formation resulting in fusion of scaroiliac and facet joints, and syndesmophyte bridging between adjacent vertebral bodies. To determine the effects of susceptibility genes on osteoclast (OC) and osteoblast (OB) development and function we have begun examining these cells differentiated from bone marrow precursors in HLA-B27 transgenic rats. The discovery that HLA-B27 misfolding in monocytes/macrophages can activate the unfolded protein response (UPR) suggests that this protein may affect cellular function beyond its canonical role as an antigen-presenting molecule. To monitor OC development, bone marrow cells were isolated from HLA-B27 transgenic and wild type (WT) rats and cultured with M-CSF to obtain OC precursors, which were then treated with M-CSF and either RANKL or TNF-&#945;to promote differentiation. TNF-&#945;dependent OC development was increased 2-3 fold (p<0.05) in HLA-B27 expressing (pre)OCs compared to WT cells as determined by numbers of OCs per culture. HLA-B27 expression was upregulated 2-fold and misfolding was increased after TNF treatment;TNF induced upregulation of BiP expression suggested UPR activation. Preliminary analyses suggest accelerated OB development in HLA-B27-expressing MSCs. IFNgamma, which is critical for OB development, induced rat MHC class I and HLA-B27 in MSCs;HLA-B27 misfolding in OBs has been documented biochemically. Our preliminary data suggest that HLA-B27 expression can accelerate OC and OB formation. Studies examining the mechanism of this effect are underway, and specificity for HLA-B27 needs to be established. UPR activation is known to be involved in OB development, and promotes the production of certain cytokines, and thus is a plausible mechanism but will need to be determined experimentally. This line of investigation will enable us to better explore the unusual juxtaposition of bone loss and bone formation in spondyloarthritis.